Identification of Cys94 as the distal ligand to the Fe(III) heme in the transcriptional regulator RcoM-2 from Burkholderia xenovorans.

The CO-responsive transcriptional regulator RcoM from Burkholderia xenovorans (BxRcoM) was recently identified as a Cys(thiolate)-ligated heme protein that undergoes a redox-mediated ligand switch; however, the Cys bound to the Fe(III) heme was not identified. To that end, we generated and purified three Cys-to-Ser variants of BxRcoM-2--C94S, C127S, and C130S--and examined their spectroscopic properties in order to identify the native Cys(thiolate) ligand. Electronic absorption, resonance Raman, and electron paramagnetic resonance (EPR) spectroscopies demonstrate that the C127S and C130S variants, like wild-type BxRcoM-2, bind a six-coordinate low-spin Fe(III) heme using a Cys/His ligation motif. In contrast, electronic absorption and resonance Raman spectra of the C94S variant are most consistent with a mixture of five-coordinate high-spin and six-coordinate low-spin Fe(III) heme, neither of which are ligated by a Cys(thiolate) ligand. The EPR spectrum of C94S is dominated by a large, axial high-spin Fe(III) signal, confirming that the native ligation motif is not maintained in this variant. Together, these data reveal that Cys(94) is the distal Fe(III) heme ligand in BxRcoM-2; by sequence alignment, Cys(94) is also implicated as the distal Fe(III) heme ligand in BxRcoM-1, another homologue found in the same organism.

Spectrophotometric titration of bimetallic metal cation binding in polyamido(amine) dendrimer templates.

Spectrophotometric titration and a binding isotherm were used to accurately assess the loading capacity of generation four polyamido(amine) (PAMAM) dendrimer templates with terminal alcohol groups (G4-OH). Preparation of bimetallic G4-OH dendrimer-encapsulated metal nanoclusters (DENs) necessitates knowledge of the precise metal-ion binding capacity. The binding of metal ions such as Pt(2+) and Pd(2+) has proven difficult to assess via UV-vis spectroscopy because the absorbance shifts associated with metal-ion binding within the dendrimer template are masked by the absorbance of the PAMAM dendrimer itself. In contrast, the binding of Cu(2+) to G4-OH PAMAM dendrimer results in a strong, distinct absorption band at 300 nm, making UV-vis spectrophotometric titration with copper straightforward. Here we use copper binding as a means to assess the number of binding sites remaining within the PAMAM G4-OH dendrimer after the complexation of a specified molar excess of Pd(2+) or Pt(2+). In addition, we use a binding isotherm to mathematically estimate the loading capacity of the dendrimer in each case. The loading capacities for M(2+) in the G4-OH dendrimer were found to be ∼16 for copper alone, ∼21 for copper combined with palladium, and ∼25 for copper combined with platinum.

Preparation and catalytic evaluation of ruthenium-nickel dendrimer encapsulated nanoparticles via intradendrimer redox displacement of nickel nanoparticles.

Ru and Ru(x)Ni(30) dendrimer encapsulated nanoparticles (DENs) were synthesized using a redox-displacement method. DEN catalytic activity for the reduction of p-nitrophenol was evaluated and found to be dependent on the ratio of metals present.

Nuclear receptors homo sapiens Rev-erbbeta and Drosophila melanogaster E75 are thiolate-ligated heme proteins which undergo redox-mediated ligand switching and bind CO and NO.

Nuclear receptors E75, which regulates development in Drosophila melanogaster, and Rev-erbbeta, which regulates circadian rhythm in humans, bind heme within their ligand binding domains (LBD). The heme-bound ligand binding domains of E75 and Rev-erbbeta were studied using electronic absorption, MCD, resonance Raman, and EPR spectroscopies. Both proteins undergo redox-dependent ligand switching and CO- and NO-induced ligand displacement. In the Fe(III) oxidation state, the nuclear receptor hemes are low spin and 6-coordinate with cysteine(thiolate) as one of the two axial heme ligands. The sixth ligand is a neutral donor, presumably histidine. When the heme is reduced to the Fe(II) oxidation state, the cysteine(thiolate) is replaced by a different neutral donor ligand, whose identity is not known. CO binds to the Fe(II) heme in both E75(LBD) and Rev-erbbeta(LBD) opposite a sixth neutral ligand, plausibly the same histidine that served as the sixth ligand in the Fe(III) state. NO binds to the heme of both proteins; however, the NO-heme is 5-coordinate in E75 and 6-coordinate in Rev-erbbeta. These nuclear receptors exhibit coordination characteristics that are similar to other known redox and gas sensors, suggesting that E75 and Rev-erbbeta may function in heme-, redox-, or gas-regulated control of cellular function.

The transcription regulator RcoM-2 from Burkholderia xenovorans is a cysteine-ligated hemoprotein that undergoes a redox-mediated ligand switch.

Spectroscopic characterization of the newly discovered heme-PAS domain sensor protein BxRcoM-2 reveals that this protein undergoes redox-dependent ligand switching and CO- and NO-induced ligand displacement. The aerobic bacterium Burkholderia xenovorans expresses two homologous heme-containing proteins that promote CO-dependent transcription in vivo. These regulators of CO metabolism, BxRcoM-1 and BxRcoM-2, are gas-responsive heme-PAS domain proteins like mammalian neuronal PAS domain protein 2 (NPAS2) and the direct oxygen sensor from Escherichia coli ( EcDos). BxRcoM-2 was studied using electronic absorption, MCD, resonance Raman, and EPR spectroscopies. In the Fe(III) oxidation state, the heme is low-spin and six-coordinate with a cysteine(thiolate) as one of the two ligands. The sixth ligand is a histidine (His (74)), which is present in all states of the protein that were studied. Reduction to the Fe(II) oxidation state results in replacement of the cysteine(thiolate) with a neutral thioether ligand, Met (104). CO and NO bind to the Fe(II) BxRcoM-2 heme opposite the histidine ligand. Thus, BxRcoM-2 employs coordination state changes similar to those known for CO-sensing CooA, with redox-dependent loss of a cysteine(thiolate) ligand and displacement of a relatively weakly bound axial ligand by the effector gas molecule. Like EcDos, the weakly bound axial ligand that is displaced is methionine.

Ferrous human cystathionine beta-synthase loses activity during enzyme assay due to a ligand switch process.

Cystathionine beta-synthase (CBS) is a pyridoxal-5'-phosphate-dependent enzyme that catalyzes the condensation of serine and homocysteine to form cystathionine. Mammalian CBS also contains a heme cofactor that has been proposed to allosterically regulate enzyme activity via the heme redox state, with FeII CBS displaying approximately half the activity of FeIII CBS in vitro. The results of this study show that human FeII CBS spontaneously loses enzyme activity over the course of a 20 min enzyme assay. Both the full-length 63-kDa and truncated 45-kDa form of CBS slowly and irreversibly lose activity upon reduction to the FeII form. Additionally, electronic absorption spectroscopy reveals that FeII CBS undergoes a heme ligand exchange to FeII CBS424 when the enzyme is incubated at 37 degrees C and pH 8.6. The addition of enzyme substrates or imidazole has a moderate effect on the rate of the ligand switch, but does not prevent conversion to the inactive species. Time-dependent spectroscopic data describing the conversion of FeII CBS to FeII CBS424 were fitted to a three-state kinetic model. The resultant rate constants were used to fit assay data and to estimate the activity of FeII CBS prior to the ligand switch. Based on this fit it appears that FeII CBS initially has the same enzyme activity as FeIII CBS, but FeII CBS loses activity as the ligand switch proceeds. The slow and irreversible loss of FeII CBS enzyme activity in vitro resembles protein denaturation, and suggests that a simple regulatory mechanism based on the heme redox state is unlikely.